Biology subjects

Rich, M. S.

Publications and source records attributed to Rich, M. S..

4 recordsLinked to original sources

bioRxiv2026

Modular gene tagging in C. elegans

Tagging a gene endogenously can identify when the gene is expressed and where the protein is localized. CRISPR is the primary tool for generating tags of endogenous genes, but it is error-prone and requires unique reagents for each gene and tag. Recombinases can insert DNA in an error-free and modular manner. Here, we tested eight recombinases for germline function in the nematode C. elegans, and introduce PhIT, a recombinase-based method for protein tagging. First, a short 39bp PhiC31 attB landing pad is inserted into the locus by CRISPR. This strain is a resource which can be used to insert a variety of modular tags. Second, tags are inserted by the integrase PhiC31, and in tandem, extraneous backbone sequences are removed by a tyrosine recombinase. Current modular tags include seven different fluorescent proteins, FLP-regulated cell-specific expression constructs, and degron tags. Importantly, tags can be inserted by genetic crosses instead of by microinjection.

genomics↗

bioRxiv2025

High-efficiency targeted integration of extrachromosomal arrays in C. elegans using PhiC31 integrase

Extrachromosomal arrays are unique chromosome-like structures created from DNA injected into the C. elegans germline. However, they are unstable unless integrated into a chromosome. Current methods for integration using X-rays or CRISPR can damage DNA and exhibit low efficiency. We demonstrate that the viral integrase PhiC31, which mediates non-mutagenic recombination between short attB and attP sequences, can be used for extremely efficient and targeted integration of arrays. Arrays were integrated by PhIAT (PhiC31-mediated Integration of Arrays of Transgenes) at attB sites on three chromosomes, including at a fluorescent landing pad. Moreover, integrations can be inserted at any arbitrary site in the genome by simultaneously co-injecting Cas9 RNP, an attB repair template, and the DNA components for the array - thereby providing sites on all six chromosomes. Single injections can integrate arrays ranging in size from 1 to 18 megabases. PhIAT makes it practical to study genomes of other organisms in the nematode; one of our strains incorporates 65% of the yeast genome at a single site in the worm genome. PhIAT will accelerate a shift from unstable extrachromosomal arrays to direct integration of arrays in C. elegans.

genetics↗

bioRxiv2022

No evidence that synonymous mutations in yeast genes are mostly deleterious

In a recent paper1, Shen et al. reported that most mutations in the coding regions of 21 yeast genes were strongly deleterious, and that the distributions of fitness effects were similar for synonymous and nonsynonymous mutations. Taken at face value, these results would conflict with well-established findings from a broad range of fields and approaches. Here, we argue that these results arose from a lack of appropriate controls for the impacts of background genetic effects in edited strains. A re-examination of the data in Shen et al. strongly suggests that it is entirely consistent with the expectation that most nonsynonymous and nearly all synonymous mutations have no detectable effects on fitness. We present analyses which show that the data is inconsistent with the proposed explanation that pervasive fitness effects of synonymous mutations arise from their effects on mRNA levels, that the sequence-based fitness assay overestimates fitness effects compared to the growth-based fitness assay, and that the observed wide fitness distributions for nonsense mutations are consistent with off-target effects or other uncontrolled sources of biological variation contributing to measured fitness. We conclude by discussing the essential controls and other experimental design considerations that are required to produce interpretable results regarding the fitness effects of mutations in large-scale screens.

genetics↗

bioRxiv2021

High-efficiency CRISPR gene editing in C. elegans using Cas9 integrated into the genome

Gene editing in C. elegans using plasmid-based CRISPR reagents requires microinjection of many animals to produce a single edit. Germline silencing of plasmid-borne Cas9 is a major cause of inefficient editing. Here, we present a set of C. elegans strains that constitutively express Cas9 in the germline from an integrated transgene. These strains markedly improve the success rate for plasmid-based CRISPR edits. For simple GFP insertions, 60 - 100% of injected animals typically produce edited progeny, depending on the target locus. Template-guided editing from an extrachromosomal array is maintained over multiple generations. We have built strains with the Cas9 transgene on multiple chromosomes. Additionally, each Cas9 locus also contains a heatshock-driven Cre recombinase for selectable marker removal and a bright fluorescence marker for easy outcrossing. These integrated Cas9 strains greatly reduce the workload for producing individual genome edits. Author SummaryGermlines have evolved specialized mechanisms to protect themselves from invasions by transposons and viruses, which create barriers to genome editing techniques. For example, transgenes are silenced in the germline of the nematode C. elegans, thereby creating a barrier to CRISPR editing by Cas9. To facilitate gene editing, we built a collection of C. elegans strains in which Cas9 is never silenced. CRISPR is significantly more efficient in these animals, decreasing the effort researchers need to expend to get edited animals. The strains are available in multiple genetic backgrounds, and contain accessory transgenes to simplify downstream genetics. Together, these strains enable efficient, low-cost genome editing in C. elegans.

genetics↗